We have set up and thoroughly investigated the use of columns of thrombin bound to Sepharose. Platelets were preincubated with 14C-serotonin and washed in a buffer containing EDTA to prevent aggregation. When the platelets are passed through short columns of thrombin/Sepharose at 37 degrees, the platelets pass through with little adhesion and secrete a varying amount of serotonin, which depends on the amount of bound thrombin. However, when platelets are passed through thrombin/Sepharose columns at 4 degrees, there is no appreciable adhesion or secretion of serotonin. If these effluent platelets are then warmed at 37 degrees they secrete an amount of serotonin equivalent to platelets on columns at 37 degrees, although we have shown that there is no thrombin present. Therefore, the platelets were stimulated by the bound thrombin at 4 degrees but do not undergo secretion if maintained at this temperature. We use the term "poised platelets" to describe the platelets in this state. We have already demonstrated that there are two proteins which are phosphorylated in poised platelets but not in the unstimulated controls. The purpose of this research is to determine whether or not the phosphorylation of these two proteins in poised platelets is accompanied by the mobilization of calcium stores into the cytosol, changes in cyclic AMP levels, or the synthesis of prostaglandins. This will be accomplished through the use of inhibitors and stimulators of these events.